The invention relates to specific biologically effective molecules on the basis of “short interfering RNA” (siRNA). After their activation said biologically effective molecules interact with the RNA of the target gene and together with special endoribonucleases they form an RNA protein complex known as “RISC” (RNA induced silencing complex). The RISC complex binds to the target mRNA and endonucleases cut the target mRNA. In this way, the gene expression is inhibited and thus the formation of target proteins is prevented.
The biologically effective molecules, which can be cell-specifically activated, can be used, for example, for combating abnormal cells and inhibiting their growth, particularly in the treatment of tumors and virus infections, in senescene-related treatments, etc. Generally, biologically effective molecules, which can be cell-specifically activated, can be used for the modulation of the gene expression of the target cells. But it is not only possible to reduce the expression of genes but also to increase it by achieving a reduction of the expression of the negative regulators of the target gene by means of the biologically active molecules.
The inhibition of the gene expression by introducing short (19-23 bp), double-stranded RNA molecules (siRNA) in eukaryotic cells, which is specific for a sequence segment of the mRNA of a target gene, was already described: Elbashir S M et al.: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature, 2001 May 24, 411(6836), 494-8; Liu Y et al.: Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 Feb. 24, 43(7), 1921-7; U.S. Pat. Nos. 5,898,031; 7,056,704).
Such molecules do not serve to inhibit the reading of a gene and the production of an mRNA but the siRNA initiates a cell's own mechanism that decomposes the target mRNA. Finally, the formation of a specific protein is inhibited without impairing the expression of further genes (post-transcriptional gene silencing).
To inhibit the expression of a gene the siRNA molecules can be directly introduced into the cell by transfection reagents and electroporation (Zhang M et al.: Downregulation enhanced green fluorescence protein gene expression by RNA interference in mammalian cells, RNA Biol. 2004 May, 1(1), 74-7; Gilmore IR et al.: Delivery strategies for siRNA-mediated gene silencing, Epub 2004 May 22., Curr Drug Deliv. 2006 Apr., 3(2), 147-5; U.S. Pat. No. 6,506,559).
The disadvantage of this method is the relative instability of the siRNA but it can be improved by chemical modifications (U.S. Pat. No. 6,107,094).
A special problem in the therapeutic application of biologically efficient molecules is an application in vivo. Methods have been introduced for such applications to stabilize the siRNA to reduce the decomposition (Morrissey et. al.: Chemical Modifications of Synthetic siRNA, Pharmaceutical Discovery, May 1, 2005), and transfection reagents, for example nanoparticles, in vivo-jetPEI™ (Polyplus), have been developed that introduce the siRNA into cells in vivo, too (Vemejoul et al.: Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer models, Cancer Research 62, 2002, 6124-31; Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A: RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo, Gene Ther 12(5), 2005, 461-6.).
Furthermore, methods have been developed to increase the transfection of cells of a target gene with siRNA in vivo (Ikeda et. al.: Ligand-Targeted Delivery of Therapeutic siRNA, Pharmaceutical Research, Vol. 23, No. 8, August 2006).
However, the administration of biologically active substances in vivo is often combined with problems due to the systemic effect. The selective introduction of these substances into target cells is not sufficiently specific. This fact is particularly disadvantageous for siRNA molecules that shall have a selective effect only in target cells. Sufficiently high cell specificity is not achieved by tissue- or cell-specifically marked transfection reagents (e.g. antibody/antigen-marked nanoparticles, TAT protein flanking, and others). Though wrong transfections occur in the prior art even when such transfection reagents are used, this does not occur when those transfection reagents are used with the deactivated molecules of the present invention.
A further known method is the deactivation of the biological effect of siRNA molecules by coupling fluorochromes and the re-transfer of said molecules to their active structure by irradiating them with light of a specific wave length (QN Nguyen et al.: Light controllable siRNAs regulate gene suppression and phenotypes in cells, Biochim Biophys Acta, 2006). This activation is initiated from the outside and is, in no way, cell-specifically directed. Consequently, the mentioned siRNA molecules have not only an effect in the corresponding target cells after their activation but, unintentionally, also in all the other transfected cells. Moreover, it is also difficult to apply this mechanism in vivo.
It is also known to deactivate the biological effect of siRNA molecules by coupling peptides that are formed so that these peptides are separated in target cells by target-cell-specific active peptidases whereas they remain inactive in non-target cells (WO002008098569A2). In this way it is possible to very selectively activate molecules on the basis of siRNA in target cells without having a negative effect of said molecules on the cell function in other cells.
Practice has proved that the link of siRNA with the peptides is not without problems and that after the separation of the one peptide or more peptides the linker that remains at the siRNA or also remaining peptide residues impair the efficacy of the siRNA in the target cells. Although the siRNA molecule is effectively deactivated during the peptide coupling, the examined linkers, and possibly also the mentioned peptide residues, that remain at the siRNA after peptide separation have a negative effect on the induction of the RNA interference thus impairing the biological efficacy of the siRNA.